Effect of a Pore-Forming Protein Derived from Flammulina velutipes on the Caco-2 Intestinal Epithelial Cell Monolayer
2004
NARAI, Asako | WATANABE, Hirohito | IWANAGA, Toshihiko | Tomita, Toshio | Shimizu, Makoto
We have previously found a transepithelial electrical resistance (TEER)-decreasing protein derived from Flammulina velutipes, which was revealed to be identical to flammutoxin (FTX) that is known as a hemolytic pore-forming protein. This protein induced a rapid decrease in TEER and parallel increase in paracellular permeability in the intestinal epithelial Caco-2 cell monolayer without any cytotoxicity. An immunoblotting analysis revealed that the FTX-induced decrease in TEER was accompanied by the formation of a high-molecular-weight complex on the surface of Caco-2 cells. Intracellular Ca²⁺ imaging showed that exposure to FTX caused a rapid Ca²⁺ influx. It was observed by electron microscopy that FTX induced swelling of microvilli and expansion of the cellular surface. Staining with fluorescent phalloidin showed a marked change to filamentous actin in the FTX-treated cells. These results suggest that TEER reduction could sensitively detect small membrane pore formation by FTX in the intestinal epithelium which causes a morphological alteration and disruption of the paracellular barrier function.
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